Abstract Proposed is the development of a simple, fast and universal strategy for incorporating radionuclides into biomolecules for applications for cancer imaging and pretargeted immunotherapy. The strategy is based on a bioconjugation reaction developed in the Fox laboratories: the reaction between tetrazines and trans- cyclooctenes (TCO). The fast kinetics of the 'tetrazine-TCO ligation' (k2 e 250,000 Lmol/s-1) enable fast reactivity at low micromolar concentrations within minutes and without an excess of either reactant. This fast reactivity provides unprecedented opportunities for protein and antibody modification and for performing reactions in vivo. This proposal involves collaborative effort from the Molecular Imaging Center (MIC) at University of Southern California (USC) and the Fox group (UD). An objective of this application is to use the tetrazine ligation to develop efficient methods for the construction of 18F labeled proteins with high specific activity for cancer imaging. Moreover, we will also develop a novel pretargeted imaging and radioimmunotherapy method that will allow for universal and straightforward tagging of monoclonal antibodies (mAbs) with an imaging/therapeutic isotope without severe radiation exposure towards normal organs. To achieve these objectives, we will first develop bioconjugation reactions with fast kinetics and robust in vivo stability based on the tetrazine-TCO ligation. As the fast kinetics of tetrazine-TCO ligation enable fast reactivity at low micromolar concentrations within minutes and without an excess of either reactant, we will transform this reaction into efficient methods that could be used for the construction of 18F labeled proteins with high specific activity. At last, we will also establish a chemical pretargeting approach that for pretargeted imaging/radioimmunotherapy of cancer based on the tetrazine-TCO ligation and anti-EphB4 antibody. In summary, in this application, we are developing efficient methods for proteins labeling with 18F, and a chemical pretargeting approach that could be used for pretargeted imaging and radioimmunotherapy of breast cancer. The feasibility of these approaches will be tested with EphB4 antibody, F(ab')2, and Fab. The success of these novel imaging and therapy approached could enable breast cancer diagnosis, make possible direct monitoring of responses to therapeutic interventions, and may significantly improve breast cancer treatment efficacy. Moreover, these newly developed approaches could have important applications in many other cancer types, and thus have a significant clinical impact on a very large number of cancer patients